1. Field of the Invention
The present invention relates generally to separation of molecular components in a fluid sample and, in particular, to an apparatus and a method for separating ion contaminants from analytes of interest in a sample fluid in an electrophoresis column.
2. Description of the Related Art
Electrophoresis is widely used for fractionation of a variety of biomolecules, including nucleic acid species, proteins, peptides, and derivatized amino acids. One electrophoretic technique which allows rapid, high resolution separation is capillary electrophoresis (CE). Typically, fused silica tubes are used in CE. These tubes are filled with a separation medium, e.g. a buffer solution or a polymer matrix such as is described in co-owned U.S. Pat. Nos. 5,164,055; 5,126,021; 5,096,554; and 5,015,350 in free-solution capillary electrophoresis or, a gel such as an agarose gel.
A CE system basically comprises an injection system, a separation system consisting of the capillary tube and buffer reservoirs in a temperature regulated compartment, a high voltage power supply, a detector, and a data processing system. The injection system generally consists of an automated mechanism that allows for both hydrodynamic (vacuum or pressure) and electrokinetic (electromigration) injection of a buffer and a sample into one and of the capillary tube. The temperature regulated environmental compartment or enclosure may contain components of the injection system as well as the capillary tube. The enclosure provides a precisely controlled environment for the capillary, ensuring that the Joule heat generated during CE is efficiently dissipated and ensuring pH stability and constant viscosity of the CE medium. The high voltage power supply typically supplies an electrophoretic driving potential of up to 30 KV between the ends of the capillary tube. The detection system is usually an ultraviolet (UV) absorbance or fluorescence based detector. The instrument data system may consist of a chart recorder or computer based data acquisition system.
To perform CE, a fluid sample volume is either hydrodynamically or electrokinetically loaded, i.e. drawn into the separation medium in one end of the capillary tube. An electric field is then applied between the ends of the tube to electrophoretically draw the charged analytes in the sample through the separation medium separating the sample components in accordance with each of the analyte's electrophoretic mobility. Molecular separation within the separation medium may be based on molecular size, in the case of nucleic acid species (which have about the same charge density), or on a combination of size, shape, and charge, in the case of proteins and peptides.
It is well known that, for many charged biopolymers of interest, e.g., single- and double-stranded DNA and sodium dodecyl sulfate (SDS)-denatured proteins, separations based on differences in electrophoretic mobilities in free solution are extremely difficult. Therefore, in order to effect electrophoretic separations of mixtures of these molecules, one has to employ a medium which exploits the frictional characteristics of these species in such a way as to enhance the molecular size dependence of electrophoretic mobility. A free solution separation medium containing an entangled polymer may be designed for this purpose. The separation medium polymer concentration and/or degree of crosslinking between the polymer molecules in such mediums may be varied to provide separation of species over a wide range of molecular weights and charges. Low viscosity entangled polymer solutions such as are disclosed in U.S. Pat. No. 5,126,021 have also been used to separate large DNA fragments and proteins.
Another alternative is to use a solid gel separation medium. For example, one preferred temperature solidified separation media is agarose gel, typically used for separating nucleic acid fragments greater than about 1000 bases, where the concentration of the agarose may vary from about 0.3%, for separating fragments in the 5-60 kilobase size range, up to about 2%, for separating fragments in the range of 100-3000 base pairs. A cross-linked polyacrylamide gel separation medium is typically used for separation of smaller size fragments, typically less than about 1000 base pairs. However, DNA restriction fragments ranging from 72 to 1353 base pairs (bp) have been separated by CE in 0.3-2.0% solutions of agarose gel at 40.degree. C. by Bocek et al, Electrophoresis 12:1059 (1991). Chen et al, Clinical Chemistry 37:14 (1991) describes separation of proteins using an agarose separation medium.
One difficulty with separations of macromolecules in capillary electrophoresis using either free solution or gel mediums is band broadening caused by spatial interference of migrating charged contaminant ions in the sample mixture with the analytes. These contaminants may be removed by desalting the sample prior to electrophoresis. Schwartz et al, Journal of Chromatography 559:267 (1991) teaches desalting, i.e. the removal of small ion contaminants, from a capillary electrophoresis column by ultrafiltration of the sample prior to detection of DNA restriction fragments by CE. This ultrafiltration technique is a separate distinct step, performed prior to introduction of the sample solution into the CE column. Such a step takes a substantial amount of time and carries with it the potential for handling errors and contamination to be introduced into the sample solution if not rigorously performed.
There is thus a need for an apparatus and method which avoids the necessity of a separate desalting step prior to loading the sample into a CE column.